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1.  Coenzyme Q0 from Antrodia cinnamomea in Submerged Cultures Induces Reactive Oxygen Species-Mediated Apoptosis in A549 Human Lung Cancer Cells 
We investigated the anticancer effects of Antrodia cinnamomea, a medicinal mushroom from Taiwan, on A549 human lung cancer cells using the ethyl acetate extract from submerged culture filtrates. Our results showed that 2,3-dimethoxy-5-methyl-1,4-benzoquinone (coenzyme Q0; CoQ0) derived from A. cinnamomea submerged culture filtrates has anticancer activity. CoQ0 treatment reduced the viability of A549, HepG2, and SW480 cancer cell lines. Furthermore, CoQ0 induced reactive oxygen species (ROS) generation and apoptosis in A549 cells, which was inhibited by the antioxidant ascorbic acid. To our knowledge, these data demonstrate for the first time that CoQ0 derived from A. cinnamomea submerged culture filtrates exerts its anticancer effect through the induction of ROS-mediated apoptosis in A549 human lung cancer cells.
PMCID: PMC4241310  PMID: 25431605
2.  Tumor Necrosis Factor-Alpha Induced by Hepatitis B Virus Core Mediating the Immune Response for Hepatitis B Viral Clearance in Mice Model 
PLoS ONE  2014;9(7):e103008.
Persistent hepatitis B viral (HBV) infection results in chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (HCC). An efficient control of virus infections requires the coordinated actions of both innate and adaptive immune responses. In order to define the role of innate immunity effectors against HBV, viral clearance was studied in a panel of immunodeficient mouse strains by the hydrodynamic injection approach. Our results demonstrate that HBV viral clearance is not changed in IFN-α/β receptor (IFNAR), RIG-I, MDA5, MYD88, NLRP3, ASC, and IL-1R knock-out mice, indicating that these innate immunity effectors are not required for HBV clearance. In contrast, HBV persists in the absence of tumor necrosis factor-alpha (TNF-α) or in mice treated with the soluble TNF receptor blocker, Etanercept. In these mice, there was an increase in PD-1-expressing CD8+ T-cells and an increase of serum HBV DNA, HBV core, and surface antigen expression as well as viral replication within the liver. Furthermore, the induction of TNF-α in clearing HBV is dependent on the HBV core, and TNF blockage eliminated HBV core-induced viral clearance effects. Finally, the intra-hepatic leukocytes (IHLs), but not the hepatocytes, are the cell source responsible for TNF-α production induced by HBcAg. These results provide evidences for TNF-α mediated innate immune mechanisms in HBV clearance and explain the mechanism of HBV reactivation during therapy with TNF blockage agents.
PMCID: PMC4105421  PMID: 25047809
3.  A novel TLR2-triggered signalling crosstalk synergistically intensifies TNF-mediated IL-6 induction 
Toll-like receptors (TLR) recognize pathogens and trigger the production of vigorous pro-inflammatory cytokines [such as tumour necrosis factor (TNF)] that induce systemic damages associated with sepsis and chronic inflammation. Cooperation between signals of TLR and TNF receptor has been demonstrated through the participation of TNF receptor 1 (TNFR) adaptors in endotoxin tolerance. Here, we identify a TLR2-mediated synergy, through a MyD88-independent crosstalk, which enhances subsequent TNF-mediated nuclear factor-kappa B activation and interleukin-6 induction. Membrane-associated adaptor MAL conduces the link between TNF receptor-associated factor 6 (TRAF6) and TNFR-associated death domain, leading to a distinctive K63-ubiquitinylated TRAF6 recruitment into TNFR complex. In summary, our results reveal a novel route of TLR signal that synergistically amplifies TNF-mediated responses, indicating an innovative target for inflammation manipulation.
PMCID: PMC4124019  PMID: 24758719
Toll-like receptor; tumour necrosis factor; signalling crosstalk; TRAF6; TRADD
4.  Ganoderma formosanum polysaccharides attenuate Th2 inflammation and airway hyperresponsiveness in a murine model of allergic asthma 
SpringerPlus  2014;3:297.
Allergic asthma is an inflammatory disease of the airways mediated by Th2 immune responses and characterized by airway hyperresponsiveness (AHR). Fungi of the genus Ganoderma are basidiomycetes that have been used in traditional Asian medicine for centuries. We recently found that PS-F2, a polysaccharide fraction purified from the submerged culture broth of Ganoderma formosanum, stimulates the activation of dendritic cells and primes a T helper 1 (Th1)-polarized adaptive immune response. This study was designed to investigate whether the Th1 adjuvant properties of PS-F2 could suppress the development of allergic asthma in a mouse model. BALB/c mice were sensitized by repeated immunization with chicken ovalbumin (OVA) and alum, followed by intranasal challenge of OVA to induce acute asthma. PS-F2 administration during the course of OVA sensitization and challenge effectively prevented AHR development, OVA-specific IgE and IgG1 production, bronchial inflammation, and Th2 cytokine production. Our data indicate that PS-F2 has a potential to be used for the prevention of allergic asthma.
Electronic supplementary material
The online version of this article (doi: 10.1186/2193-1801-3-297) contains supplementary material, which is available to authorized users.
PMCID: PMC4072879  PMID: 25019045
Airway hyperresponsiveness; Allergic asthma; Ganoderma formosanum; Immunomodulation; Polysaccharides
5.  Mapping the Distinctive Populations of Lymphatic Endothelial Cells in Different Zones of Human Lymph Nodes 
PLoS ONE  2014;9(4):e94781.
The lymphatic sinuses in human lymph nodes (LNs) are crucial to LN function yet their structure remains poorly defined. Much of our current knowledge of lymphatic sinuses derives from rodent models, however human LNs differ substantially in their sinus structure, most notably due to the presence of trabeculae and trabecular lymphatic sinuses that rodent LNs lack. Lymphatic sinuses are bounded and traversed by lymphatic endothelial cells (LECs). A better understanding of LECs in human LNs is likely to improve our understanding of the regulation of cell trafficking within LNs, now an important therapeutic target, as well as disease processes that involve lymphatic sinuses. We therefore sought to map all the LECs within human LNs using multicolor immunofluorescence microscopy to visualize the distribution of a range of putative markers. PROX1 was the only marker that uniquely identified the LECs lining and traversing all the sinuses in human LNs. In contrast, LYVE1 and STAB2 were only expressed by LECs in the paracortical and medullary sinuses in the vast majority of LNs studied, whilst the subcapsular and trabecular sinuses lacked these molecules. These data highlight the existence of at least two distinctive populations of LECs within human LNs. Of the other LEC markers, we confirmed VEGFR3 was not specific for LECs, and CD144 and CD31 stained both LECs and blood vascular endothelial cells (BECs); in contrast, CD59 and CD105 stained BECs but not LECs. We also showed that antigen-presenting cells (APCs) in the sinuses could be clearly distinguished from LECs by their expression of CD169, and their lack of expression of PROX1 and STAB2, or endothelial markers such as CD144. However, both LECs and sinus APCs were stained with DCN46, an antibody commonly used to detect CD209.
PMCID: PMC3986404  PMID: 24733110
6.  Extracellular polysaccharides produced by Ganoderma formosanum stimulate macrophage activation via multiple pattern-recognition receptors 
The fungus of Ganoderma is a traditional medicine in Asia with a variety of pharmacological functions including anti-cancer activities. We have purified an extracellular heteropolysaccharide fraction, PS-F2, from the submerged mycelia culture of G. formosanum and shown that PS-F2 exhibits immunostimulatory activities. In this study, we investigated the molecular mechanisms of immunostimulation by PS-F2.
PS-F2-stimulated TNF-α production in macrophages was significantly reduced in the presence of blocking antibodies for Dectin-1 and complement receptor 3 (CR3), laminarin, or piceatannol (a spleen tyrosine kinase inhibitor), suggesting that PS-F2 recognition by macrophages is mediated by Dectin-1 and CR3 receptors. In addition, the stimulatory effect of PS-F2 was attenuated in the bone marrow-derived macrophages from C3H/HeJ mice which lack functional Toll-like receptor 4 (TLR4). PS-F2 stimulation triggered the phosphorylation of mitogen-activated protein kinases JNK, p38, and ERK, as well as the nuclear translocation of NF-κB, which all played essential roles in activating TNF-α expression.
Our results indicate that the extracellular polysaccharides produced by G. formosanum stimulate macrophages via the engagement of multiple pattern-recognition receptors including Dectin-1, CR3 and TLR4, resulting in the activation of Syk, JNK, p38, ERK, and NK-κB and the production of TNF-α.
PMCID: PMC3495220  PMID: 22883599
Ganoderma formosanum; Polysaccharide; Immunostimulatory; Macrophage; Pattern-recognition receptor
7.  Preparation of a Porous Composite Film for the Fabrication of a Hydrogen Peroxide Sensor 
Sensors (Basel, Switzerland)  2011;11(6):5873-5885.
A series of dopant-type polyaniline-polyacrylic acid composite (PAn-PAA) films with porous structures were prepared and developed for an enzyme-free hydrogen peroxide (H2O2) sensor. The composite films were highly electroactive in a neutral environment as compared to polyaniline (PAn). In addition, the carboxyl group of the PAA was found to react with H2O2 to form peroxy acid groups, and the peroxy acid could further oxidize the imine structure of PAn to form N-oxides. The N-oxides reverted to their original form via electrochemical reduction and increased the reduction current. Based on this result, PAn-PAA was used to modify a gold electrode (PAn-PAA/Au) as a working electrode for the non-enzymatic detection of H2O2. The characteristics of the proposed sensors could be tuned by the PAA/PAn molar ratio. Blending PAA with PAn enhanced the surface area, electrocatalytic activity, and conductivity of these sensors. Under optimal conditions, the linear concentration range of the H2O2 sensor was 0.04 to 12 mM with a sensitivity of 417.5 μA/mM-cm2. This enzyme-free H2O2 sensor also exhibited a rapid response time, excellent stability, and high selectivity.
PMCID: PMC3231461  PMID: 22163932
polyaniline; polyacrylic acid; enzyme-free; hydrogen peroxide sensor
8.  Human CD141+ (BDCA-3)+ dendritic cells (DCs) represent a unique myeloid DC subset that cross-presents necrotic cell antigens 
The Journal of Experimental Medicine  2010;207(6):1247-1260.
The characterization of human dendritic cell (DC) subsets is essential for the design of new vaccines. We report the first detailed functional analysis of the human CD141+ DC subset. CD141+ DCs are found in human lymph nodes, bone marrow, tonsil, and blood, and the latter proved to be the best source of highly purified cells for functional analysis. They are characterized by high expression of toll-like receptor 3, production of IL-12p70 and IFN-β, and superior capacity to induce T helper 1 cell responses, when compared with the more commonly studied CD1c+ DC subset. Polyinosine-polycytidylic acid (poly I:C)–activated CD141+ DCs have a superior capacity to cross-present soluble protein antigen (Ag) to CD8+ cytotoxic T lymphocytes than poly I:C–activated CD1c+ DCs. Importantly, CD141+ DCs, but not CD1c+ DCs, were endowed with the capacity to cross-present viral Ag after their uptake of necrotic virus-infected cells. These findings establish the CD141+ DC subset as an important functionally distinct human DC subtype with characteristics similar to those of the mouse CD8α+ DC subset. The data demonstrate a role for CD141+ DCs in the induction of cytotoxic T lymphocyte responses and suggest that they may be the most relevant targets for vaccination against cancers, viruses, and other pathogens.
PMCID: PMC2882828  PMID: 20479116
9.  Uric acid promotes an acute inflammatory response to sterile cell death in mice 
The Journal of Clinical Investigation  2010;120(6):1939-1949.
Necrosis stimulates inflammation, and this response is medically relevant because it contributes to the pathogenesis of a number of diseases. It is thought that necrosis stimulates inflammation because dying cells release proinflammatory molecules that are recognized by the immune system. However, relatively little is known about the molecular identity of these molecules and their contribution to responses in vivo. Here, we investigated the role of uric acid in the inflammatory response to necrotic cells in mice. We found that dead cells not only released intracellular stores of uric acid but also produced it in large amounts postmortem as nucleic acids were degraded. Using newly developed Tg mice that have reduced levels of uric acid either intracellularly and/or extracellularly, we found that uric acid depletion substantially reduces the cell death–induced inflammatory response. Similar results were obtained with pharmacological treatments that reduced uric acid levels either by blocking its synthesis or hydrolyzing it in the extracellular fluids. Importantly, uric acid depletion selectively inhibited the inflammatory response to dying cells but not to microbial molecules or sterile irritant particles. Collectively, our data identify uric acid as a proinflammatory molecule released from dying cells that contributes significantly to the cell death–induced inflammatory responses in vivo.
PMCID: PMC2877935  PMID: 20501947
10.  Neutrophil Influx and Chemokine Production during the Early Phases of the Antitumor Response to the Vascular Disrupting Agent DMXAA (ASA404)1 
Neoplasia (New York, N.Y.)  2009;11(8):793-803.
5,6-Dimethylxanthenone-4-acetic acid (DMXAA) acts through tumor vascular disruption and cytokine production and is the first of its class to enter phase 3 trials. We characterized leukocytes and cytokines in murine Colon 38 tumors before and after DMXAA treatment. Tumor mass declined 50% 24 hours after DMXAA administration, but the leukocyte count per gram of tumor increased threefold owing to a large influx of Ly6G+CD11b+F4/80- cells with the morphology of neutrophils. However, B and T lymphocytes, natural killer cells, and macrophages in the tumor all decreased in numbers. Seven chemokines were substantially induced in the tumor, spleen, and serum 4 hours after DMXAA administration. Using cultured spleen cell subpopulations, CD11b+ cells (largely monocytes and macrophages) were shown to be the primary producers of tumor necrosis factor α, interleukin 6 (IL-6), and macrophage inflammatory 1α (MIP-1α). CD49b+ natural killer cells produced IP-10, whereas CD45R+ B lymphocytes produced regulated upon activation normal T cell express sequence. T lymphocytes were not major producers of cytokines in the response to DMXAA. Murine peripheral blood leukocytes (PBLs) produced a similar panel of cytokines in culture to that detected in mouse serum after DMXAA treatment. Cytokines in human PBL cultures were subsequently measured with the aim of identifying potential serum markers of the human response to DMXAA. IP-10 (P < .001), monocyte chemoattractant protein 1 (P < .001), and sCD40L (P < .01) were decreased, whereas IL-8 (P < .001) and MIP-1α (P = .03) were increased in DMXAA-treated compared with untreated PBL cultures from a group of 12 donors.
PMCID: PMC2713591  PMID: 19649209
11.  Orally Administered Amyloidophilic Compound Is Effective in Prolonging the Incubation Periods of Animals Cerebrally Infected with Prion Diseases in a Prion Strain-Dependent Manner▿  
Journal of Virology  2007;81(23):12889-12898.
The establishment of effective therapeutic interventions for prion diseases is necessary. We report on a newly developed amyloidophilic compound that displays therapeutic efficacy when administered orally. This compound inhibited abnormal prion protein formation in prion-infected neuroblastoma cells in a prion strain-dependent manner: effectively for RML prion and marginally for 22L prion and Fukuoka-1 prion. When the highest dose (0.2% [wt/wt] in feed) was given orally to cerebrally RML prion-inoculated mice from inoculation until the terminal stage of disease, it extended the incubation periods by 2.3 times compared to the control. The compound exerted therapeutic efficacy in a prion strain-dependent manner such as that observed in the cell culture study: most effective for RML prion, less effective for 22L prion or Fukuoka-1 prion, and marginally effective for 263K prion. Its effectiveness depended on an earlier start of administration. The glycoform pattern of the abnormal prion protein in the treated mice was modified and showed predominance of the diglycosylated form, which resembled that of 263K prion, suggesting that diglycosylated forms of abnormal prion protein might be least sensitive or resistant to the compound. The mechanism of the prion strain-dependent effectiveness needs to be elucidated and managed. Nevertheless, the identification of an orally available amyloidophilic chemical encourages the pursuit of chemotherapy for prion diseases.
PMCID: PMC2169081  PMID: 17881452
12.  MyD88-dependent IL-1 receptor signaling is essential for gouty inflammation stimulated by monosodium urate crystals 
The Journal of Clinical Investigation  2006;116(8):2262-2271.
While it is known that monosodium urate (MSU) crystals cause the disease gout, the mechanism by which these crystals stimulate this inflammatory condition has not been clear. Here we find that the Toll/IL-1R (TIR) signal transduction adaptor myeloid differentiation primary response protein 88 (MyD88) is required for acute gouty inflammation. In contrast, other TIR adaptor molecules, TIRAP/Mal, TRIF, and TRAM, are not required for this process. The MyD88-dependent TLR1, -2, -4, -6, -7, -9, and -11 and IL-18 receptor (IL-18R) are not essential for MSU-induced inflammation. Moreover, MSU does not stimulate HEK cells expressing TLR1–11 to activate NF-κB. In contrast, mice deficient in the MyD88-dependent IL-1R showed reduced inflammatory responses, similar to those observed in MyD88-deficient mice. Similarly, mice treated with IL-1 neutralizing antibodies also showed reduced MSU-induced inflammation, demonstrating that IL-1 production and IL-1R activation play essential roles in MSU-triggered inflammation. IL-1R deficiency in bone marrow–derived cells did not affect the inflammatory response; however, it was required in non–bone marrow–derived cells. These results indicate that IL-1 is essential for the MSU-induced inflammatory response and that the requirement of MyD88 in this process is primarily through its function as an adaptor molecule in the IL-1R signaling pathway.
PMCID: PMC1523415  PMID: 16886064
13.  Murine Coronavirus Nonstructural Protein p28 Arrests Cell Cycle in G0/G1 Phase 
Journal of Virology  2004;78(19):10410-10419.
Murine coronavirus mouse hepatitis virus (MHV) gene 1 encodes several nonstructural proteins. The functions are unknown for most of these nonstructural proteins, including p28, which is encoded at the 5′ end of the MHV genome. Transient expression of cloned p28 in several different cultured cells inhibited cell growth, indicating that p28 expression suppressed cell proliferation. Expressed p28 was exclusively localized in the cytoplasm. Cell cycle analysis by flow cytometry demonstrated that p28 expression induced G0/G1 cell cycle arrest. Characterization of various cellular proteins that are involved in regulating cell cycle progression demonstrated that p28 expression resulted in an accumulation of hypophosphorylated retinoblastoma protein (pRb), tumor suppressor p53, and cyclin-dependent kinase (Cdk) inhibitor p21Cip1. Expression of p28 did not alter the amount of p53 transcripts yet increased the amount of p21Cip1 transcripts, suggesting that p28 expression increased p53 stability and that p21Cip1 was transcriptionally activated in a p53-dependent manner. Our present data suggest the following model of p28-induced G0/G1 cell cycle arrest. Expressed cytoplasmic p28 induces the stabilization of p53, and accumulated p53 causes transcriptional upregulation of p21Cip1. The increased amount of p21Cip1 suppresses cyclin E/Cdk2 activity, resulting in the inhibition of pRb hyperphosphorylation. Accumulation of hypophosphorylated pRb thus prevents cell cycle progression from G0/G1 to S phase.
PMCID: PMC516409  PMID: 15367607
14.  Murine Coronavirus Replication Induces Cell Cycle Arrest in G0/G1 Phase 
Journal of Virology  2004;78(11):5658-5669.
Mouse hepatitis virus (MHV) replication in actively growing DBT and 17Cl-1 cells resulted in the inhibition of host cellular DNA synthesis and the accumulation of infected cells in the G0/G1 phase of the cell cycle. UV-irradiated MHV failed to inhibit host cellular DNA synthesis. MHV infection in quiescent 17Cl-1 cells that had been synchronized in the G0 phase by serum deprivation prevented infected cells from entering the S phase after serum stimulation. MHV replication inhibited hyperphosphorylation of the retinoblastoma protein (pRb), the event that is necessary for cell cycle progression through late G1 and into the S phase. While the amounts of the cellular cyclin-dependent kinase (Cdk) inhibitors p21Cip1, p27Kip1, and p16INK4a did not change in infected cells, MHV infection in asynchronous cultures induced a clear reduction in the amounts of Cdk4 and G1 cyclins (cyclins D1, D2, D3, and E) in both DBT and 17Cl-1 cells and a reduction in Cdk6 levels in 17Cl-1 cells. Infection also resulted in a decrease in Cdk2 activity in both cell lines. MHV infection in quiescent 17Cl-1 cells prevented normal increases in Cdk4, Cdk6, cyclin D1, and cyclin D3 levels after serum stimulation. The amounts of cyclin D2 and cyclin E were not increased significantly after serum stimulation in mock-infected cells, whereas they were decreased in MHV-infected cells, suggesting the possibility that MHV infection may induce cyclin D2 and cyclin E degradation. Our data suggested that a reduction in the amounts of G1 cyclin-Cdk complexes in MHV-infected cells led to a reduction in Cdk activities and insufficient hyperphosphorylation of pRb, resulting in inhibition of the cell cycle in the G0/G1 phase.
PMCID: PMC415820  PMID: 15140963
15.  Nucleocapsid-Independent Specific Viral RNA Packaging via Viral Envelope Protein and Viral RNA Signal 
Journal of Virology  2003;77(5):2922-2927.
For any of the enveloped RNA viruses studied to date, recognition of a specific RNA packaging signal by the virus's nucleocapsid (N) protein is the first step described in the process of viral RNA packaging. In the murine coronavirus a selective interaction between the viral transmembrane envelope protein M and the viral ribonucleoprotein complex, composed of N protein and viral RNA containing a short cis-acting RNA element, the packaging signal, determines the selective RNA packaging into virus particles. In this report we show that expressed coronavirus envelope protein M specifically interacted with coexpressed noncoronavirus RNA transcripts containing the short viral packaging signal in the absence of coronavirus N protein. Furthermore, this M protein-packaging signal interaction led to specific packaging of the packaging signal-containing RNA transcripts into coronavirus-like particles in the absence of N protein. These findings not only highlight a novel RNA packaging mechanism for an enveloped virus, where the specific RNA packaging can occur without the core or N protein, but also point to a new, biologically important general model of precise and selective interaction between transmembrane proteins and specific RNA elements.
PMCID: PMC149775  PMID: 12584316
16.  Induction of Apoptosis in Murine Coronavirus-Infected Cultured Cells and Demonstration of E Protein as an Apoptosis Inducer 
Journal of Virology  1999;73(9):7853-7859.
We demonstrated that infection of 17Cl-1 cells with the murine coronavirus mouse hepatitis virus (MHV) induced caspase-dependent apoptosis. MHV-infected DBT cells did not show apoptotic changes, indicating that apoptosis was not a universal mechanism of cell death in MHV-infected cells. Expression of MHV structural proteins by recombinant vaccinia viruses showed that expression of MHV E protein induced apoptosis in DBT cells, whereas expression of other MHV structural proteins, including S protein, M protein, N protein, and hemagglutinin-esterase protein, failed to induce apoptosis. MHV E protein-mediated apoptosis was suppressed by a high level of Bcl-2 oncogene expression. Our data showed that MHV E protein is a multifunctional protein; in addition to its known function in coronavirus envelope formation, it also induces apoptosis.
PMCID: PMC104316  PMID: 10438879

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